Fig. 6: miR-30d enhances tumorigenesis and metastasis in vivo.

a Tumor volume measured at the indicated time points after orthotopic xenograft of MDA-MB-231–Luc control (CTRL, n = 8) or miR-30d decoy (dy-30d, n = 9) cells in SCID mice. p value: p = 0.00255667, p = 0.00021304, p = 0.00000072, and p = 0.00000029, respectively, at 7, 14, 21, and 28 days. b Representative images of immunohistochemical analyses (IHC) of markers of Golgi (GM130), collagen deposition (Picro Sirius red), and CAFs (α-SMA) in serial sections from primary tumors in (a) (CTRL, n = 8; dy-30d, n = 9). Lower panels show higher magnification of upper panels’ insets. c, d Quantification of immunohistochemistry markers analyzed in (c). e In vivo luciferase quantification of lung metastases from mice in (a). (a) and (b) report the mean +/− SEM with CTRL (n = 8) and decoy-30d (n = 9) (p value: p = 0.0011 and p = 0.0265, respectively, at 28 and 35 days). f Whole-body in vivo luciferase quantification at the indicated time points of control or miR-30d decoy MDA-MB-231–Luc cells injected intravenously in SCID mice (CTRL n = 6, dy-30d n = 9, p = 0.0011134, p = 0.0000023, respectively, at 21 and 28 days). g Ex vivo luciferase quantification at 28 days in lungs explanted from mice in (d) (CTRL n = 6, dy-30d n = 9, p = 0.0004). h Schematic representation of the mouse conditioning experiment. SCID mice were injected with medium conditioned by MDA-MB-231 cells transfected with control or miR-30d inhibitor (IH-30d), before performing orthotopic xenografts. Metastases were evaluated after resection of primary tumors. i Confidence lines showing whole-body growth of metastases at the indicated days after surgical removal of primary tumors (Wilcoxon rank-sum test; n = 8 for each group). j Representative images of immunohistochemical (IHC) analyses for markers of proliferation (Ki67), collagen deposition (Picro Sirius red), CAFs (α-SMA), and endothelial cells (CD31), in metastases from serial lung sections of mice in (h) and (i) (n = 8 for each group). Lower panels show magnification of insets in the upper panels. k–n Quantification of immunohistochemistry markers reported in (j). All graphs report single data points, mean +/− SEM (p value: *p < 0.05, ***p < 0.001, ****p < 0.0001 by two-tailed unpaired Student’s t-test). Source data are provided as Source data file.